This invention relates to processes for controlling the sialic acid content of glycoproteins produced in mammalian cell culture. The invention provides processes for increasing and decreasing the sialic acid content of glycoproteins produced by mammalian cell culture. The invention further relates to processes for producing tumor necrosis factor receptor (TNFR)-immunoglobulin (Ig) chimeras as well as novel TNFR1-IgG1 preparations and their uses in the diagnosis and treatment of various inflammatory and immune disorders.
Differences in glycosylation patterns of recombinantly produced glycoproteins have recently been the topic of much attention in the scientific community as recombinant proteins produced as probable prophylactics and therapeutics approach the clinic. The oligosaccharide side chains of the glycoproteins affect the protein""s function (Wittwer A., and Howard, S. C. (1990) Biochem. 29:4175-4180) and the intramolecular interaction between portions of the glycoprotein resulting in the conformation and presented three dimensional surface of the glycoprotein (Hart, (1992) Curr. Op. Cell Biol., 4:1017-1023; Goochee, et al., (1991) Bio/Technology, 9:1347-1355; Parekh, R. B., (1991) Curr. Op. Struct. Biol., 1:750-754). Oligosaccharides may also serve to target a given polypeptide to certain structures based upon specific cellular carbohydrate receptors (Bevilacqua, M. P. and Nelson, R. M., (1993) J. Clin. Invest. 91:379-387; Nelson, R. M., et al., (1993) J. Clin. Invest. 91:1157-1166, Norgard, K. E. et al., (1993) Proc. Natl. Acad. Sci. USA 90:1068-1072; Imai, Y. et al., (1993) Nature 361:555-557). The terminal sialic acid component of the glycoprotein oligosaccharide side chain affects absorption, serum half life, and clearance from the serum, as well as the physical, chemical and immunogenic properties of the glycoprotein(Parekh, R. B., supra; Varki, A., (1993) Glycobiology 3:97-100; Paulson, J. (1989), TIBS, 14:272-276; Goochee, et al., (1991) Biotechnology 9:1347-1355; Kobata, A, (1992) Eur. J. Biochem. 209:483-501). It is therefore important to maintain the sialic acid content of glycoproteins, particularly of those proteins intended for use as therapeutics.
Much attention has been paid to the factors which affect glycosylation during recombinant protein production such as growth mode (adherent or suspension), fetal bovine serum in media formulation, culture density, oxygenation, pH, purification schemes and the like (Werner, R. and Noe, W. (1993), Drug Res. 43:1134-1249; Hayter et al., (1992) Biotech. and Bioeng. 39:327-335; Borys et al., (1994) Biotech and Bioeng. 43:505-514; Borys et al., (1993) Bio/technology 11:720-724; Hearing et al., (1989) J. Cell Biol. 108:339-353; Goochee et al., in Frontiers in Bioprocessing II, Todd et al., eds (1992) American Chemical Society pp.199-240; U.S. Pat. No. 5,096,816; Chotigeat, W., (1994) Cytotech. 15:217-221). Several groups have investigated the process parameters that surround the production of recombinant proteins and especially the effect of media composition in the production of recombinant proteins (Park et al., (1992) Biotech. Bioeng. 40:686-696; Cox and McClure, (1983) In Vitro, 19:1-6; Mizutani et al., (1992) Biochem. Biophys. Res. Comm. 187:664-669; Le Gros et al., (1985) Lymph. Res. 4(3):221-227).
Addition of alkanoic acids such as butyric acid are known to effect the transient expression of foreign DNA in recombinant cell culture (Prasad, and Sinha, (1976) In Vitro, 12:125-132; Japanese Patent Application No. 62-48935; Japanese Patent Application No. 55-150440; Klehr et al., (19:92) Biochem. 31:3222-3229; Gorman, and Howard, (1983) Nucleic acid Res. 11:7631-7648). However, sodium butyrate has a range of effects on gene expression across various cell lines and media compositions (D""Anna et al., (1980) Biochem. 19:2656-2671; Hagopian, H. K., (1977) Cell 12:855-860) and protein production (Milhaud (1980) J. Cell. Physiol. 104:163-170; U. K. Patent Application No. GB 2 122 207 A) suggesting that butyrate may modify gene expression (Yuan et al., (1985) J. Biol. Chem. 3778-3783) or inhibit the expression of certain genes (Yuan et al., supra).
European Patent No. 0 239 292 B1 describes a process for the enhanced production of protein in the presence of an alkanoic acid or a salt thereof such as butyric acid. The publication, however, provides little guidance in selecting appropriate concentrations of the additive and further does not address the effect of the additive on protein glycosylation. Others have described that the addition of low levels (0-1.5 mM) of sodium butyrate to cell culture production medium to increase cell specific productivity leads to concomitant increases in acidic glycoforms (corresponding to increased sialic acid content) of the recombinant protein produced (Chotigeat, et al., (1994) Cytotech. 15:217-221).
Several groups have looked at the effects of osmolality on cell growth and polypeptide production (Ozturk and Palsson (1991) Biotech. and Bioeng. 37:989-993; Stubblefield et al., (1960) Cancer Research, 20:1646-1655; Garcia-Perez et al., (1989) Journal of Biological Chemistry, 264(28):16815-16821; Miner et al., (1981) Invasion Metastasis, 1:158-174; GB 2,251,249; EP 481, 791; U.S. Pat. No. 5,151,359; U.S. Pat. No. 4,724,206; U.S. Pat. No. 5,122,469; and WO 89/04867). Various osmolality ranges for cell growth or polypeptide production have been proposed. Generally, the osmolality of the cell culture medium is increased via the addition of NaCl or amino acids. Environmental stresses such as increased salt concentrations lead, in some instances, to increased cell product production. The notion that increased expression of mammalian protein products can be achieved in mammalian cell cultures through solute stress, e.g., the addition of salt, lactic acid, ammonia to the culture media has been reported (International Publication No. WO 89/04867). These stresses are generally growth inhibitory but favor cell specific productivity.
Others have discussed the effect of glucose concentration on cell growth and/or polypeptide production in recombinant cell culture. See, for example, Park et al., (1992) Biotechnology and Bioengineering, 40:686-696; Huang et al., (1991) Journal of Biotechnology, 18:161-162; EP 387, 840; Reuveny et al., (1986) Journal of Immunological Methods, 86:53-59; Fine et al., (1976) In Vitro, 12(10):693-701; Dircks et al., (1987) Exp. Eye Res., 44:951-958; Mizutani et al., (1992) Biochemical and Biophysical Research Communications, 187(2):664-669; Sugiura, (1992) Biotechnology and Bioengineering, 39:953-959; WO 88/01643; Graf et al., (1989) DECHEMA Biotechnol. Conf., 3:615-618; Japanese Patent Application No. JP 1-101882; U.S. Pat. No. 3,926,723; WO 87/00195; and Fleischaker, Jr., Ph.D. Thesis, Massachusetts Institute of Technology, pp. 196-229 (June 1982). However, the previous studies have not studied the effect of various process parameters on the sialic acid content of the mature protein, a factor in glycoprotein production that is tantamount to clinical success.
The present invention provides for processes for controlling the content of sialic acid of glycoproteins produced by mammalian cell culture.
The present inventors have discovered that certain mammalian cell culture process parameters affect cell specific productivity as well as the extent and type of glycosylation of the proteins produced. More particularly, the present inventors have found that certain factors which enhance cell specific productivity have an inverse effect on the sialic acid content of the produced protein. The present inventors have therefore devised various cell culture processes to enrich particular glycoforms of glycoproteins produced in mammalian cell culture.
Accordingly, the invention provides for a process for controlling the sialic acid content of a glycoprotein produced by mammalian cell culture. According to this aspect of the invention, varying the production rate of the glycoprotein in the production phase of the cell culture leads to variations in the sialic acid content of the mature glycoprotel in. More particularly, an increase in cell specific productivity during the glycoprotein production phase results in a decrease in sialic acid content of the mature protein. Conversely, a decrease in cell specific productivity results in an increase in sialic acid content in the mature protein.
The present invention provides, in a particular embodiment, for varying the cell specific productivity of a mammalian host cell during the protein production phase of mammalian cell culture by controlling factors which affect cell specific productivity. According to one aspect of the invention, the concentration of factors which enhance DNA transcription are controlled. In another embodiment cell specific productivity is controlled by maintaining the osmolality of the cell culture within certain margins. According to the invention, any of the foregoing parameters are controlled, alone or in combination, to affect the mature glycoprotein sialic acid content.
In a particular embodiment of the present invention, the factor which enhances DNA transcription is an alkanoic acid or salt thereof such as sodium butyrate at a concentration of about 0.1 mM to about 20 mM. According to a second aspect of the invention, the osmolality of the cell culture is maintained between about 250-600 mOsm. In a further aspect, the temperature of the cell culture is controlled between about 30xc2x0 C. and 37xc2x0 C.
In a preferred embodiment, the invention provides for a process for increasing the sialic acid content of the mature glycoprotein produced by mammalian cell culture comprising maintaining a lower cell specific productivity by controlling any or all of the above identified process parameters, optionally together with other parameters known in the art. According to this aspect of the present invention, culturing the host cell at a concentration of the alkanoic acid or salt thereof of about 0.1 mM to about 6 mM, and optionally together with maintaining the osmolality of the cell culture at about 300-450 mOsm produces a protein with an increased sialic acid content.
In a further preferred embodiment, the invention provides for a process for decreasing the sialic acid content of the mature glycoprotein produced by mammalian cell culture comprising increasing cell specific productivity of the cell culture. The cell specific productivity is increased, in a preferred embodiment, by providing a cell culture process which comprises any of, culturing the host cell at a concentration of an alkanoic acid or salt thereof of about 6 mM to about 12 mM; and, maintaining the osmolality of the cell culture at about 450-600 mOsm.
The invention further provides, in a particular embodiment, for a cell culture process with three phases of cell culture. The invention therefore provides a process for controlling the sialic acid content of a glycoprotein produced by mammalian cell culture comprising the steps of culturing a host cell which expresses the protein in a growth phase for a period of time and under such conditions that cell growth is maximized. According to this aspect of the present invention, the growth phase is followed by a transition phase in which cell culture parameters for the desired sialic acid content of the mature glycoprotein are selected and engaged. The transition phase is followed by a production phase of the cell culture wherein parameters selected in the transition phase are maintained and glycoprotein product is produced and harvested. Varying the cell specific productivity of the production phase of the cell culture by adding an alkanoic acid or a salt thereof to the cell culture at a concentration of about 0.1 mM to about 20 mM and engaging an osmolality of the cell culture at about between 250 and 600 mOsm, optionally in combination with one another during the transition phase produces a protein with differing amounts of sialic acid.
In a further preferred embodiment, the present invention provides a process for controlling the amount of sialic acid present in a soluble type 1 tumor necrosis factor receptor (TNFR1)-immunoglobulin G1(IgG1) chimeric protein. The present inventors have discovered that, under certain conditions of production, novel TNFR1-IgG1 glycoform preparations may be obtained which exhibit the desirable properties of prolonged clearance from the blood while retaining significant functional activity. A long functional half-life permits simplified, bolus-dose administration and contributes to in vivo potency of the glycoprotein produced allowing for lower dose forms of the glycoprotein.
According to this aspect of the present invention, a TNFR1-IgG1 glycoprotein molecule is produced that contains increased sialic acid residues. The cell culture parameters for the production phase of the TNFR1-IgG1 are selected to obtain the desired sialic acid content. In a preferred embodiment, the sodium butyrate is present in the production phase at a concentration of about 0.1 to about 6 mM and the osmolality is maintained at about 300-450 mOsm. In a more preferred aspect the sodium butyrate concentration is about 1 mM and the osmolality is maintained at about 350-400 mOsm.
In yet another embodiment, the present invention provides for a preparation of TNFR1-IgG1 glycoprotein produced by the process of the present invention. According to this aspect of the invention a preparation is provided comprising TNFR1-IgG1 in which the range of pI of the preparation is between about 5.5 and 7.5. Further provided is a TNFR1-IgG1 preparation having a molar ratio of sialic acid to protein of about 4 to about 7 and especially about 5 to about 6. In yet another aspect, the TNFR1-IgG1 preparation has about 1 to about 2 moles of exposed N-acetylglucosamine residues per mole of protein. In a further aspect, the preparation has a molar ratio of sialic acid to N-acetylglucosamine of about 0.35 to about 0.5 and more preferably about 0.4 to about 0.45.
The present invention also provides a therapeutic composition comprising the above preparation useful in the treatment of TNF-mediated pathologic conditions.